Project Abstract Pain affects more Americans than heart disease, cancer and diabetes combined, making pain the major reason for Americans to seek healthcare services. Approximately a quarter of the population experiences craniofacial pain mediated by the trigeminal neurosensory system. Many are more common in women, such as migraine and temporomandibular pain disorder. While it is clear that trigeminal pain is a major health issue for both men and women, women are not well represented in pain research. One theory for the prevalence of trigeminal pain disorders in females is hormone modulation of trigeminal pain mechanisms. Clinical and basic science research indicates that gonadal hormones may increase and/or decrease pain; however the link between gonadal hormones and trigeminal pain is unknown. One trigeminal pain mechanism that may be under hormonal influence is the peripheral serotonergic system, which has been highly implicated in trigeminal system mediated pain. The neurotransmitter serotonin (5HT) is a proinflammatory mediator in the periphery and an increase in 5HT levels has been associated with high estrogen. Our recent studies suggest that peripheral 5HT evokes pain via trigeminal nociceptors expressing the transient receptor potential V1 channel (TRPV1), activated by capsaicin and noxious heat. TRPV1 activation triggers the release of the proinflammatory calcitonin gene-related peptide (CGRP) and plays a critical role in inflammatory mediator sensitization of nociceptors. The overall approach of the present R15 grant application is to identify whether gonadal hormones modulate the effects of 5HT on the TRPV1 population of trigeminal nociceptors in females. Experiments outlined are designed to test the hypothesis that gonadal hormones modulate serotonergic potentiation of TRPV1-expressing trigeminal sensory neurons in rodents and humans. Specific Aim 1 will determine if gonadal hormone fluctuations predictably alter serotonergic potentiation of rat trigeminal nociception using established rodent trigeminal sensitivity behavioral assays and neuroanatomical techniques. Specific Aim 2 will identify whether gonadal hormones enhance serotonergic potentiation of TRPV1 in rat trigeminal sensory neurons using electrophysiology and primary neuronal culture methodologies. Specific Aim 3 will test if 5HT2A receptor expression and potentiation of capsaicin-evoked CGRP release in human dental pulp varies across the menstrual cycle using western blot and superfusion methodology in extracted human dental pulp. The results of the proposed research are critical to understanding how hormones may regulate pronociceptive activity of peripheral serotonin at both rat and human trigeminal nociceptors. This knowledge will provide insight on peripheral craniofacial pain mechanisms in women, which may provide translational insight for sex-based recommendations and novel therapeutics for women. Our preliminary data provide strong support for the hypothesis and the results will have a significant translational impact. Importantly, this proposal is designed to provide a rigorous biomedical research environment for undergraduate and graduate students.